Up to now, there have been no primary standards available for use with glycated protein assays. Controls and calibrators do exist, but they are only assigned values by either:
1) assaying secondary standards alongside the control and extrapolating the value for that control or 2) initially and arbitrarily assigning a value to a control and attempting to match subsequent control values to this initial control value by running known and unknown controls side by side and extrapolating. Thus the lack of a primary standard is a source of error in these assays. An example of using a secondary standard to calibrate an assay is the fructosamine assay as taught by Baker in U.S. Pat. Nos. 4,642,295 and 4,645,742 or Rosenthal in EPO Patent Application 85/307,206. In these patents, 1-deoxy-1-morpholinofructose (DMF) is used in an aqueous solution of a protein to standardize the reaction. DMF is considered a secondary standard because it reacts in a similar fashion to glycated protein in the fructosamine assay. DMF cannot be considered a primary standard because it is not a glycated protein. An example of a control value being arbitrarily assigned in the hemoglobin A.sub.1 (Hb A.sub.1) assay is that of Acuff et al.'s U.S. Pat. No. 4,238,196. In this assay, columns which measure glycated hemoglobin (Hb A.sub.1) are used to initially assign a value to a pooled hemoglobin control, and subsequent batches of both columns and controls are matched to the value of this initial control.
The major difficulty involved in preparing a primary protein standard is the unavailability of a starting material that is not glycated. The major sources of starting material for controls are human or animal blood products including serum, plasma, red cells (hemoglobin), and albumin. These protein sources are all glycated to some extent. One approach to obtaining nonglycated protein would be to separate the non-glycated protein from the glycated protein using chromatography. The principal disadvantages of this approach are:
(a) Proteins may be denatured to some extent during chromatography.
(b) Large amounts of protein are needed and this would require the use of large columns which are difficult and time-consuming to run.
(c) The resulting nonglycated product may still be glycated to some degree.
The non-availability of a primary standard for measuring glycated protein has made calibration of the various assays difficult indeed. This is probably best illustrated in the fructosamine assay. Phillipou et al., Clinical Chem. 34 1561 (1988), point out that DMF, on reaction with nitro blue tetrazolium chloride (NBT), gives a different visible spectrum clearly dissimilar from that obtained for protein or plasma samples. Addition of protein to the DMF solution before introduction of the NBT reagent changed DMF's spectral characteristics to mimic those observed for serum samples. The use of DMF/protein calibrators has several practical limitations and Phillipou et al. above, referenced 15 publications addressing the question of assay standardization and problems thereof. One particularly thorny problem is that the addition of protein to DMF solutions adds an unknown amount of fructosamine to each calibrator. Another problem is that there is no true zero standard because all available proteins are glycated. Phillipou et al. also point out that the use of DMF as a calibrant causes overestimation of the amount of fructosamine present in a plasma sample.